PROBLEM: Aging is the most important risk factor for Alzheimer's disease (AD), which represents the most common cause of dementia in our country. The disease, for which there is no currently available treatment, is becoming increasingly prevalent among our aging veteran population. PRELIMINARY DATA: Autophagy is an essential component of the cell degrading machinery. It helps dispose of large toxic protein aggregates that form within the secretory pathway and in the cytosol. Malfunction of autophagy and disruption of proteostasis contributes to the progression of many chronic diseases. In addition, many chronic degenerative diseases are characterized by the aberrant accumulation of toxic protein aggregates. Compelling data indicate that increased levels of autophagy can be beneficial in mouse models of diseases characterized by increased accumulation of toxic protein aggregates, including AD. As such, improving normal proteostatic mechanisms is an active target for biomedical research. N?- lysine acetylation was initially thought to occur only in the cytoplasm and nucleus. However, in 2007 we discovered that the endoplasmic reticulum (ER) is also able to acetylate newly-synthesized polypeptides. Since then, we have successfully identified the entire biochemical machinery responsible for ER-acetylation and generated relevant animal models. The machinery includes AT-1, which translocates acetyl-CoA from the cytosol to the ER lumen, and ATase1/ATase2, two acetyltransferases that carry out the enzymatic reaction within the ER lumen. We discovered that the ER acetylation machinery maintains the homeostatic balance of two essential and intimately related functions of the ER: (i) ?positive? selection of correctly folded nascent polypeptides and (ii) tight regulation of autophagy/reticulophagy. Mice with reduced influx of acetyl- CoA into the ER (AT-1S113R/+) display excessive induction of autophagy and a block of the secretory pathway while mice with increased influx (AT-1 Tg and AT-1 sTg) display increased efficiency of the secretory pathway and a block of normal reticulophagy. In both cases, lack of homeostatic balance leads to drastic phenotypes. Relevant to this proposal is also the fact that a dysfunctional ER acetylation machinery has been linked to aging and AD. Consistently, haploinsufficiency of AT-1 or biochemical inhibition of the ATases was able to rescue the AD-like phenotype in the mouse. HYPOTHESIS: Our general hypothesis is that the ER acetylation machinery ensures protein homeostasis. Deregulation of this cross-talk impacts both aging and AD. STUDY DESIGN: Specific Aim 1 will identify novel structure-based ATase1 and ATase2 inhibitors to prevent AD. This Aim will take advantage of new structural information that we have collected on the ATases and new structure-based inhibitors that we have recently identified. Relevant structural biochemistry, in vitro and ex vivo analysis, and pre-formulation/formulation development of these novel compounds have already been completed. They will now be tested in two mouse models of AD. This Aim will also take advantage of ATase1-/- and ATase2-/- mice, recently generated in our laboratory to determine whether targeting only one ATase is sufficient to rescue AD neuropathology in the mouse. Specific Aim 2 will identify the molecular mechanism(s) that provides specificity to the proteostatic functions of the ER acetylation machinery. Under this Aim we report the identification of a novel ER-based acetyltransferase that appears to play an important role in the regulation of autophagy/reticulophagy down-stream of the ER acetylation machinery. This Aim is highly mechanistic and includes a combination of structural biochemistry, molecular biology and in vitro/ex vivo analysis.